银杏叶提取物对大鼠急性脊髓损伤后神经功能恢复的作用及其机制

目的:观察银杏叶提取物(EGb)对大鼠实验性脊髓损伤后组织结构及运动功能恢复的作用,探讨其对急性脊髓损伤的作用机制。方法:120只SD雄性大鼠,随机分为假手术组(A组)、损伤对照组(B组)、甲基强的松龙(MP)治疗组(C组)和EGb治疗组(D组),每组30只。B、C、D组用Allen′s法以50g·cm致伤大鼠T9脊髓制作损伤模型,B组为单纯脊髓损伤,不给药;C组为脊髓损伤后30min内,由腹腔注入MP30mg/kg;D组为术后至处死前每天腹腔给予EGb17.5mg/kg;A组只打开T9椎板,不打击脊髓,不给药。术后24h、3d、5d、7d、14d对大鼠进行脊髓运动功能(BBB)评分。于术后24h、3d、5d、7d、14d处死动物(n=6),取T9节段脊髓,切片苏木素-伊红(HE)染色观察脊髓大体组织结构变化,用免疫组织化学方法检测B细胞淋巴瘤/白血病基因-2(Bcl-2)和B细胞淋巴瘤/白血病基因伴随蛋白x(Bax)在脊髓前角运动神经元中的表达变化情况。结果:各时间点B、C、D组大鼠脊髓运动功能(BBB)评分均显著低于A组(P<0.01),伤后7d、14d时C、D组评分显著高于B组(P<0.05),各时间点C组与D组评分比较无统计学意义(P>0.05)。HE染色C组和D组大鼠脊髓损伤区较B组坏死程度轻、形成囊腔少,A组正常;1周后D组较C组片状出血灶少,神经细胞肿胀不明显。各时间点B、C、D组大鼠损伤脊髓前角运动神经元中Bcl-2阳性细胞数均显著高于A组(P<0.01),C、D组显著高于B组(P<0.01),7d、14d时D组显著高于C组(P<0.05);各时间点B、C、D组大鼠损伤脊髓前角运动神经元中Bax阳性细胞数均显著高于A组(P<0.01),C、D组显著低于B组(P<0.01),7d、14d时D组显著低于C组(P<0.01)。结论:EGb可能通过抑制Bax表达、提高Bcl-2表达,抑制脊髓损伤后神经元凋亡,在运动功能恢复、损伤脊髓组织保护上发挥其有益作用,1周后EGb仍能抑制脊髓损害后的继发性损伤。

The function of the extracts of Ginkgo biloba on rats spinal cord injury and its mechanism

Objective:To observe whether the extracts of Ginkgo biloba(EGb) can enhance the functional and structural recovery of the injured spinal cord of rats and to study the mechanism of EGb on spinal cord injury.Method:120 SD rats were randomly divided into four groups:sham group(group A),control group(group B),methylprednisolone(MP) treated group(group C) and EGb treated group(group D),30 rats for each group.The modified Allen method was adopted to make the models of T9 spinal cord injury.For group B the spinal cord were only injured,but not injection.For group C,MP(30mg/kg) was injected immediately by intraperitoneal injection.In group D,EGb was delivered 17.5mg/kg/d through intraperitoneally.BBB score was recorded at 24h,3d,5d,7d,14d respectively.The rats were sacrificed at 24h,3d,5d,7d,14d postinjury(n=6) and T9 spinal cord specimen was obtained,then the change of spinal cord was observed by HE stain through light microscope and the expression levels of B-cell lymphoma/leukemia gene-2(Bcl-2) and Bcl-associated x protein(Bax) protein in spinal cord were detected by immunohistochemistry assay.Result:The BBB score in group B,C and D were significantly lower than that in group A at each time point(P<0.01),the score of group C and D was significantly higher than that of group B(P<0.05) at 7 and 14 day time points,but group C and D showed no statistically significant differernce(P>0.05) at each time point.HE stain showed the spinal cord of group C and D has fewer necrosis and the vacuole area in spinal cord than that of group B,while the spinal cord was normal in group A,group D has fewer hemorrhage and swelling of nerve cells than that of group C.The number of Bcl-2 positive of motor neuron in group B,C and D were significantly higher compared with those in group A(P<0.01),however,the numbers of group C and D was significantly higher than that of group B(P<0.01) at each time point,group D was significantly higher than that of group C at 7 and 14 day time points(P<0.05).The number of Bax positive of motor neuron in group B,C and D were significantly higher compared with those in group A(P<0.01),however,the numbers of group C and D was significantly lower than that of group B(P<0.01) at each time point,group D was significantly lower than that of group C at 7 and 14 day time points(P<0.01).Conclusion:EGb can inhibit abnormal expression of Bax protein,and increase the expression of Bcl-2 protein as to inhibit apoptosis of neuron after spinal cord injury.The efficacy of EGb was proved by the recovery of functional locomotion and the protection of the injured spinal cord,EGb appear that can inhibit secondary lesion one week after injury.